A critical step in the characterization of large genomes, including that of humans, has been the cloning of large chromosomal fragments. This has been fulfilled through the development of artificial chromosomes in the yeast S. cerevisiae (YACs) and has led to the large scale physical map of the human genome. In order to improve fidelity of human DNA cloning in yeast we developed an alternative approach for isolating DNA as large circular and linear YACs that omits the in vitro ligation step. The approach is based on transformation-associated recombination (TAR) between a repeat within transformed human DNA (such as an Alu) and a human repeat sequence(s) on a co-transformed linearized plasmid. The circular YACs have several features resembling bacterial artificial chromosomes, BACs, in that they are stable and they can be easily isolated. More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human/rodent hybrid cell lines and radiation hybrids. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats. The level of enrichment of human DNA was nearly 3,000-fold. A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human-rodent chimeras were detected among YACs generated by TAR cloning. Thus, TAR cloning greatly expands the usefulness of YACs in that it provides the possibility for direct, highly specific cloning of DNA fragments through recombination. It provides the opportunity for the simple isolation of specific chromosome sequences and it is likely to lead to the isolation of gene families, and possibly single copy genes.